Final QuickVet Multicenter Report 290610-final - smb.dk 11 - Final...QuickVet values to the results on the ACL TOP for cats, the study indicates that there is a an ... algorithm (old)

Confidential Page 1 BWI 29-06-2010

FACULTY OF LIFE SCIENCES

UNIVERSITY OF COPENHAGEN

BO WIINBERG, DVM, PHD

Assistant Professor

Internal Medicine & ECC

DEPARTMENT OF SMALL ANIMAL

CLINICAL SCIENCES

DYRLÆGEVEJ 16

1870 FREDERIKSBERG C

TELEPHONE 3528 2930

DIRECT 3533 2924

FAX 3528 2929

[email protected]

WWW.DYREHOSPITALET.KU.DK

REF: SMB

BEDES OPLYST VED HENV.

Frederiksberg, June 29th

2010

Final internal report on results of a clinical multicenter

performance study of the QuickVet/Vspro 10LM

PI LIFE: Bo Wiinberg

CI KU: Annemarie T Kristensen, Anette Urbrand Martinsen

PI SMB: Niels Kristian Bau Madsen

CI SMB: Ole Kring

This report is a summary of the results from a data analysis of samples collected in a clinical

multicenter study on the performance of the QuickVet/VSpro 10LM combo coagulation

catridge.

Aims of the study The study was carried out in an effort to evaluate the clinical performance of the Quick-

Vet/VSpro 10LM system. The study has included:

• Correlation to current gold standard in veterinary coagulation testing (ACL TOP 500)

• Establishment of sensitivity and specificity of the system in relation to clinical signs

of systemic coagulopathy at various cut-off points (ROC curve analyses)

• Intra-assay and inter-assay analytical variation

• Determination of guideline reference values

Materials and Methods The study has primarily been carried out at the following locations:

• Department of Small Animal Clinical Sciences, Faculty of Life Sciences, University of

Copenhagen, Frederiksberg, Denmark

• Department of Clinical Sciences, Cummings Scholl of Veterinary Medicine, Tufts Uni-

versity, North Grafton, Massachusetts, USA

• Regions Djursjukhuset Helsingborg, Helsingborg, Sweden

Clinically relevant citrated blood samples were collected from dogs and cats at the clinicians’

discretion. One blood sample was used for duplicate QuickVet/VSpro analyses and the other

spun down and plasma stored for PT and aPTT analyses in Copenhagen. At the initiation of

the study it was planned to include 100 samples from each species.

The analyses are based on samples collected from 165 sick dogs and 35 sick cats. 51 dog

samples and 7 cat samples were excluded from the final data analyses due to lack of docu-

mentation, protocol not adhered to, but the majority due to a cartridge production error.

Positive predictive value, negative predictive value, sensitivity and specificity and cut-off for

optimal detection have been calculated based on clinical data.

Samples from 25 healthy dogs and 13 cats have been used to establish guideline reference

values. We recommend that data from at least healthy 40 dogs and cats is collected in order

to establish a more robust reference range.

GraphPad Prism 5.0 and MedCalc were used for statistical analyses.


Performance –Agreement

Correlation Analyses Dog

The analyses are based on samples collected from 165 sick dogs. As for correlation of the

QuickVet/VSpro values to the results on the ACL TOP for dogs, the study indicates that there

is a an acceptable correlation for PT (Pearson correlation = 0,72 and Spearman = 0,47) and

slightly better for aPTT (Pearson correlation = 0,93 and Spearman correlation = 0,59). The

Spearman correlation is more precise for small sample sizes of data that is not normally dis-

tributed. A D'Agostino & Pearson omnibus normality test has shown that the data is not nor-

mally distributed and therefore the Spearman test is most precise.

ACL TOP


ACL TOP


Comments Correlation Plots for Canine PT and aPTT:

It is evident from the scatterplot (Dog aPTT QuickVet/VSpro new algorithm) that 10 dogs had

prolonged aPTT on the ACL TOP, but normal aPTT on the QuickVet/VSpro system. The dogs

are indicated on the scatterplot with a blue broken line.

From a review of the patient files it is evident that most of these dogs had either a systemic

disease with a systemic inflammatory response (gastroenteritis, poisoning, acute renal failure

etc.) or they had intracavitary bleeding (thorax/abdomen /pericardium) due to tumour rup-

ture.

There is no obvious common denominator in the files of these dogs, which could explain why

they had prolonged aPTT on the ACL TOP, but normal aPTT on the QuickVet/VSpro system.

With regard to the dogs with a systemic inflammatory response, it is well known that such

patients can have prolonged aPTT without having a systemic coagulopathy. The prolonged

aPTT in these cases are often caused by the inflammatory response.

With regard to the dogs with neoplasia and intracavitary bleeding, there are several likely

explanations for the prolonged aPTT on the ACL TOP:

1. These dogs all had a localized mechanic bleeding that was so severe, that it could

cause a minor systemic coagulopathy, which was detected by the ACL TOP, but not

the QV system.

2. Dogs with severe hemoabdomen often receive large amounts of fluids during the ini-

tial resuscitation. Such therapy could have a transient effect on the clotting times,

wich the ACL TOP is sensitive enough to pick up.

3. Another common denominator for these cases is that they all had neoplasia and

therefore likely a systemic inflammatory response, which may be the cause of the

prolonged aPTT.

A common feature for all the dogs aPTT on the ACL TOP, but normal aPTT on the Quick-

Vet/VSpro system is that they were all suffering from diseases that could predispose to de-

velopment of disseminated intravascular coagulation DIC. These dogs could therefore be in

an early state of DIC. However such patients usually have concomitant prolongation of PT,

low platelet count and high d-dimers and there is not enough data to support that these dogs

were all suffering from DIC.

Correlation to Bleeding - a Clinical Example Though the data analyses shows that there is not a perfect correlation between the two sys-

tems for dogs and cats in the normal and slightly elevated range, the results from a dog with

severe coagulopathy correlate very well with the clinical signs and effect of therapy.

The highest measurements recorded are pre- and post transfusion from a dog that had in-

gested rat poison. The dog bled pre transfusion, but not post transfusion. The results from

this dog indicate that both the aPTT and PT tests are sensitive towards detection of severe

coagulopathy secondary to vitamin K antagonist ingestion, with a PT of 27 seconds (with the

new algorithm, failure with the old algorithm) and aPTT 250 seconds. The tests also show that

PT normalized to 16 seconds post transfusion while aPTT was 116 seconds after transfusion.

These findings correlate well with what was found on the ACL TOP 500.

Data from dog with coagulopathy secondary to ingestion of rat poison

Pre transfusion Post transfusion

PT QV (<17sec) 27 sec 16 sec

PT TOP (<8,5 sec) Out of range 8,2 sec

aPTT QV (<106 sec) 252 sec 116,8 sec

aPTT TOP (<12,5 sec) 160 sec 18,5 sec


Correlation Analyses Cat

The analyses are based on samples collected from 35 sick cats. As for correlation of the

QuickVet values to the results on the ACL TOP for cats, the study indicates that there is a an

acceptable correlation for PT (Pearson correlation = 0,47 and Spearman = 0,41) and much

better for aPTT (Pearson correlation = 0,76 and Spearman correlation = 0,82). The Spearman

correlation is more precise for small sample sizes of data that is not normally distributed. A

D'Agostino & Pearson omnibus normality test has shown that the data is not normally dis-

tributed and therefore the Spearman test is most precise.

ACL TOP


ACL TOP


Analytical Performance The analytical performance of the assays has been evaluated by performing duplicate meas-

urements on samples from 25 healthy dogs and 13 cats. Coefficients of Variation (CVs) were

determined using the arithmetic mean and variance estimate, based on the difference be-

tween the duplicate samples.

The analytical variation (CVa) of the assays on dog are:

P T N E W

A L G O R I T H M P T O L D

A L G O R I T H M A P T T N E W

A L G O R I T H M A P T T O L D

A L G O R I T H M

I N T R A I N T R A I N T R A I N T R A

1 , 2 % 2 , 2 % 2 , 8 % 2 , 6 %

For comparison, the imprecision data (Intra-assay CVs) for the ACL TOP PT and aPTT are ap-

proximately:

Comment: it is evident that the repeatability performance of the QuickVet is excellent and

room for improvement is therefore very limited. The CV’s for cat are comparable (3,4% for

PT and 3,8% for aPTT). The no clot and error 8 problems which were seen with the previous

algorithm (old) have not been an issue with the new software.

Guideline reference ranges Samples from clinically 25 healthy dogs and 13 cats have been used to establish guideline

reference values. We recommend that data from at least healthy 40 dogs and cats is collected

in order to establish a more robust reference range.

Based on the limited data the range of measurements (minimum-maximum) will most likely

give a reference range closest to the truth (highlight in green below). Alternatively the refer-

ence range can be based on statistical propability indicated by the 95% and 99% CI below,

however the limited amount of data gives a high standard deviation, which causes the refer-

ence ranges to be quiet broad if calculated this way.

P T A P T T

I N T R A I N T R A

1 , 6 % 3 , 2 %


Setting the Optimum Cut-off for Detection of Systemic Coagulopathy with

Clinical Signs

In order to establish optimum cut-off values and the effect these will have on sensitivity and

specificity for detection of systemic coagulopathy, ROC curve analyses have been performed.

These analyses indicate that placing the cut-off values at the upper end of the reference

range will make both the PT and the aPTT assays very specific and less sensitive.

In a specialised lab, such as the one at the Department of Small Animal Clinical Sciences, LIFE,

KU, we have set up our assays so that they are very sensitive, which gives a number of false

positives. However our assays are used as screening assays and we have other assays such as

thromboelastography to confirm coagulopathy in patients with a slightly prolonged PT or

aPTT.

Based on the ROC curves on the following pages and the results from clinically healthy cats

and dogs we recommend that the upper end of the reference value be placed around:

PT dog < 18 sec

PT cat < 18 sec

aPTT dog < 106 sec

aPTT cat < 120 sec

ROC Curve Analyses Explanations:

-PV = Negative predictive value

+PV = Positive predictive value

-LR = Negative likelihood ratio

-+LR = Positive likelihood ratio

Red asterix signifies highest combined sensitivity and specificity


ROC Curve Analyses aPTT QuickVet/VSpro


ROC Curve Analyses PT QuickVet/VSpro


ROC Curve Analyses ACL TOP 500 for Comparison:


ROC Curve Analyses ACL TOP 500 for Comparison:


Conclusion

The aPTT and PT QV/VsPro 10LM assays are performing well compared to the comparable

ACL TOP assays under these clinical conditions. The LM10 system has impressive repeatability

and good usability, with acceptable agreement to the ACL TOP system. Agree-

ment/correlation analyses do not reveal how the QuickVet system performs with regard to

detection of bleeding or risk of bleeding and therefore is of limited value.

Therefore the 10 LM assays have also been analysed for correlation to clinical signs in order

to establish optimal cut-off values for detection of systemic coagulopathy (ROC Curve Analy-

ses). It must be stressed that several studies have shown that the PT and aPTT assays do not

always perform well in this regard and it is our recommendation that samples from dogs with

clinical signs of systemic coagulopathy are continually collected, in order to improve the data

quality. Knock out studies can likely be performed to determine how the system performs on

samples with single factor (FVII, FVIII and FIX) deficiencies.

Guideline reference ranges have been established in this study. A stronger guideline refer-

ence interval than the one which has been established at this point will require 40-50 dogs

and cats, however true reference ranges will require at least 200 dogs and cats.

Though the performance does not equal that of the the ACL TOP 500, , it is our opinion that

the QV/VsPro 10LM system performs well as a simple patient near PT and aPTT test. The

machine is easy to operate, the tests are easy to run and the results easy to interpret.

Both the PT and aPTT assay have a high specificity with regard to detection of marked sys-

temic coagulopathy. Although these are opposite characteristics compared to most plasma

based assays, which have a high sensitivity, we are of the opinion that the high specificity

should be viewed as a positive attribute in a primary practice setting.

A high specificity will ensure that there are few false positives and also that those animals

which have a prolonged PT/aPTT on the QV/VsPro system have a clinically significant coagu-

lopathy. In other words, the tests can be used to confirm that clinical signs of bleeding are

due to a marked systemic coagulopathy..

Sincerely

Bo Wiinberg & Annemarie T. Kristensen


Notes:

Documents

Final QuickVet Multicenter Report 290610-final - smb.dk 11 - Final...QuickVet values to the results on the ACL TOP for cats, the study indicates that there is a an ... algorithm (old)